Although not exclusively, this invention is mainly related to a procedure for the simultaneous quantification of the major types of human lymphocytes (T, B, and NK cells) and their subsets (CD3+/CD4+, CD3+/CD8+, etc) up to a total of 12 lymphoid subpopulations by means of a quick, cost effective, sensitive and specific single measurement capable of being analyzed in a flow cytometer equipped with a single laser for research or diagnostic, prognostic and therapeutic purposes.
Lymphocytes are the cells responsible for immunosurveillance as well as for the specificity of immune defense in humans. The immunophenotyping of lymphoid subpopulations by combining the use of monoclonal antibodies and flow cytometry provides objective and sensitive measurements of both the distribution of the lymphoid subpopulations and their intensity of positivity for a certain antigen.
Both types of information have rapidly attracted the attention of professionals from different areas of Medicine such as Cell Biology, Immunology and Hematology, as the measurement of different lymphoid subsets has proved to be of great diagnostic and prognostic value in different pathologic conditions.
Of all these applications, one whose usefulness should be pointed out is that of the enumeration of CD4+ T-cells in those individuals who display serum antibodies against the human immunodeficiency virus (HIV) responsible for the acquired immunodeficiency syndrome (AIDS). This measurement represents at present one of the most important prognostic criteria that should be taken into account when analyzing these patients' progress.
The clinical relevance of this kind of measurement has largely contributed to the fact that over the past few years there has been an enormous increase in the need for appropiate quality control procedures when analyzing lymphoid subpopulations by flow cytometry in order to eliminate technical artifacts that may lead to erroneous results.
As a result of these studies dealing with the standardization of lymphoid subset measurement using flow cytometry, several research groups, scientific associations and public institutions have published documents with recommendations and guidelines for everyone to follow when immunophenotyping human lymphocytes by means of flow cytometry. These documents recognize the need for systematic identification of: 1) the proportion and purity of the lymphocytes analyzed, i.e., the proportion of lymphocytes not included in the analysis as well as the proportion of the events analyzed that correspond to lymphocytes; and 2) the total number of T, B and NK cells as well as CD3+/CD4+ and CD3+/CD8+ T-cell subsets. This means that a minimal panel of monoclonal antibodies must be used which in turn means that a high number of measurements must be carried out per sample.
As an example, in 1992 the CDC (Center for Disease Control, USA) recommended the use of five different measurements combining two monoclonal antibodies with different fluorochromes for the enumeration of CD4+ T-cells in HIV+ patients in order to fulfill the two objectives mentioned above:
1) CD45/CD14 for the study of the purity and the proportion of lymphocytes analyzed.
2) CD3/CD19 for the identification of the total number of T-lymphocytes (CD3+/CD19-) and the total number of B-lymphocytes (CD3-/CD19+).
3) CD3/CD56 for the characterization of NK-cells (CD56+/CD3-).
4) CD3/CD4 for the identification of CD3+/CD4+ T-cells which are related to helper/induction functions within the immune system.
5) CD3/CD8 for the identification of CD3+/CD8+ T-cells witch are associated with suppresor/cytotoxic immunologic functions.
Until now, no procedure has been described that allows direct and simultaneous analysis of the total number of T, B, NK, CD3+/CD4+ and CD3+/CD8+ populations in a single measurement, eliminating the need to perform up to five different measurements using ten monoclonal antibodies in double-staining combinations.